The research proposed here is directed toward an understanding of the molecular mechanism of general genetic recombination. This goal is approached through the study of the structure of special sites in DNA promoting a high rate of recombination in their vicinity, and through the identification and study of the activity of the protein which recognizes these sites. The Chi recombinational hotspots of bacteriophage lambda and its host Escherichia coli will be studied in the proposed research. We have already determined the nucleotide sequence encompassing one of these Chi sites in lambda and we have isolated mutations in this site which inactivate it. We will continue this analysis by determining the sequence around other Chi sites in lambda and in E. coli. In addition, we will determine the nucleotide changes of the mutations we have isolated in one of these sites. Mutations in other Chi sites will be isolated and sequenced in the proposed research. Comparison of these sequences will define the recognition sequence for the recombinational protein whose activity is stimulated by these hotspots. We will seek a new class of special sites which act in conjunction with the Chi sites. These sites, in the proper configuration with Chi, activate Chi sites. These activator sites will be sought through a combination of genetic and gene-splicing techniques. The protein which recognizes Chi will be sought by biochemical and genetic approaches. Nucleases and DNA-binding proteins with a preference for Chi DNA over non-Chi DNA will be sought. Mutants of E. coli in which Chi is non-functional will be obtained through enrichment and mass screening procedures. The protein altered or missing in these mutants will be identified, and its activity will be determined. Studies of the protein and its activity on Chi will lead to an understanding of the molecular mechanism by which Chi sites stimulate general recombination.